Abstract INTRODUCTION: The utility of photosensitizers (PS), such as 5-Aminolevulinic acid (5-ALA), has been limited to fluorescence-guided surgery (FGS) and photodynamic therapy. The downstream metabolism of 5-ALA results in tumor-specific, highly selective accumulation of PpIX, an endogenous porphyrin, in tumor cells. We have previously reported evidence of PpIX in EVs derived from the plasma of glioblastoma (GBM) patients. Here, we characterize the tumor-specific nature of PpIX fluorescent EVs through quantification of their cargo, demonstrating their utility in the advancement of liquid biopsy for GBM diagnosis and management. METHODS: Fluorescence activated cell sorting was adapted for nanoparticle isolation and PpIX fluorescent EVs were sorted from plasma collected from patients (N = 8) diagnosed with GBM, undergoing FGS. RNA cargo was extracted and quantified via low input RNA-Seq. Matching patient tumor and total plasma EVs along with healthy control plasma EVs (N = 8) were sequenced in parallel. SUMMARY: Through sequencing analysis, we have demonstrated a distinct landscape of long RNAs (mRNA and lncRNA) in PpIX EVs, with isolation of this EV population resulting in an enrichment of lncRNAs. Considering the emerging diagnostic and prognostic potential of lncRNAs in GBM, exploration of such EVs provides valuable insight into the pathobiology of the tumor of origin. On a broader scale, gene expression analysis of PpIX EV cargo demonstrates at least a ten-fold enrichment of genes that are otherwise buried in the heterogeneous plasma EV background. Furthermore, downstream KEGG orthological analysis of PpIX EV cargo has revealed alignment of 78% of the significant pathways to processes implicated in cancer development and progression. We have identified a panel of genes unique to PpIX EVs, and reflective of the native tumor, that are implicated in GBM pathogenesis including GREM1, MAP4K4, and STAG2. Validation via quantitative and digital PCR of total plasma EVs from patient and healthy cohorts (N = 10, respectively) have yielded similar expression levels, as reflected in sequencing gene expression. Isolation of PpIX EVs, however, insinuates enriched genetic detection. CONCLUSION: In summary, this preliminary cohort of GBM patients has demonstrated the utility of 5-ALA induced PpIX fluorescence outside of its original intentions: facilitating tumor-specific EV identification for the progression of liquid biopsy. These findings are an inviting breakthrough in the development of individualized disease detection and monitoring. Citation Format: Tiffaney Hsia, Anudeep Yekula, Syeda M. Batool, Bob S. Carter, Leonora Balaj. Novel approach for glioblastoma characterization via tumor specific extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5175.